The objectives of this project are to construct viral mutants unable to be reactivated from latency, in order to incorporate these mutations into the vaccine strains developed in Project 1. Several lines of research will indicate which regions of the genome can be mutated to reduce reactivation. The specific objectives are as follows: The first objective stems from the observation that there is a delay from the time of initial human infection to the time of the first recurrence (median of approximately 120 days). An animal model that mimics this characteristic of human infection has been developed and will be utilized to determine whether this delay is due to the time necessary for the viral genome to be amplified to a copy number necessary for reactivation. The second objective stems from the results of current studies showing that the HSV genome contains two origins of DNA replication utilized by host polymerases (oriH1 and oriH2). Sequences required for replication of these origins will be identified. Viruses containing mutations in one or both of these origins will be assayed for their ability to be reactivated from late y. The third objective is based on reports from numerous other laboratories indicating that the sequences around the L-S junction of the virus that are transcribed during latency (LAT-transcriptional domain) play a role in reactivation. Systematic mutations of this region will be incorporated into the virus and the recombinant viruses will be assayed for their ability to be reactivated from latency. The results of these studies will be joined with the results of Project 1 and Project 3 in order to construct a vaccine strain with the lowest possible chance of reactivation from latent infection.